We propose to develop ML265, a small molecule PKM2 (M2 isoform of pyruvate kinase) activator, into a new targeted therapeutic for Acute Myeloid Leukemia (AML). The majority (91%) of AML cases is diagnosed in adults, and incidence increases significantly among elderly population. The outcomes for adults with AML are poor. Although chemotherapy results in high rates of remission, the majority of patients relapse, with an overall 5 year survival only 40-50% for younger patients and a median overall survival less than 1 year for older patients. The persistence of leukemia stem cells (LSCs) following chemotherapy is a major factor contributing to clinical relapse. Therefore, there is an urgent need to develop novel therapeutics that specifically target LSCs in AML patients. Pyruvate kinase catalyzes the last step of glycolysis. Its M2 isoform, PKM2, is highly expressed in tumors of all kinds, including AML cells, but with low pyruvate kinase activity. PKM2 occurs in tetramer (active) and dimer (inactive) forms; a high tetramer-to-dimer ratio leads to energy production, while a low ratio channels metabolites into synthetic processes. ML265 potently activates human PKM2 in vitro, induces PKM2 tetramer formation, and significantly reduces aerobic glycolysis in cancer cells. In addition, ML265 also increases reactive oxygen species (ROS) concentrations and induces oxidative stress in cancer cells. PKM2 is abundantly expressed (up to 11.3x of control) in many AML cell lines and primary AML patient samples suggesting all subtypes of AML could benefit from ML265 treatment. ROS is important for the maintenance of malignant LSCs in AML. Significantly lower ROS level is observed in LSCs-like CD34+ cells compared to more mature CD34- cells. In human primary AML samples, ML265 increases ROS level and significantly reduces the colony formation of LSCs. In AML transgenic (MLL-AF9) mouse model, deletion of PKM2 with expression of PKM1 (a situation similar to ML265 treatment) reduces the levels of metabolic intermediates important for biosynthesis and impairs leukemic cells in blood and bone marrow without perturbing normal hematopoietic stem cells (HSCs). ML265 is well tolerated in repeat dose toxicity studies in mice suggesting a greater than 5X margin of safety based on an in vitro efficacious concentration in human primary AML samples. Our SBIR Phase I goal is to establish the pre-clinical proof-of-concept and demonstrate that ML265 selectively targets LSCs and complements standard therapy in AML patient derived xenograft (PDX) mouse. The specific aims proposed in this proposal include: (1) Determine the optimal concentration of ML265 via in vitro viability assays. At this concentration, ML265 not only strongly reduces the survival of phenotypically described LSCs, but is also well tolerated by normal HSCs. (2) We will investigate whether ML265 could selectively target LSCs using AML PDX model. We will treat the AML PDX mice with ML265 for 4 weeks, followed by a secondary serial transplantation to validate killing of LSCs. (3) Using AML PDX mouse, we will further determine whether ML265 could complement current standard induction chemotherapy and extend the life span of chemotherapy-treated AML mouse. We will treat the AML PDX mice with ML265, induction chemotherapy, or ML265 in combination with induction chemotherapy. The dosing of ML265 will last for 8 weeks, mice will be monitored for survival and signs of leukemia. Demonstrating that ML265 selectively targets LSCs and complements standard therapy in AML will justify Phase II research and open development of a new way to attack one of world's most deadly cancers.